Guanyl nucleotide binding proteins (GNPs) are critical in the regulation of receptor mediated events. The stimulatory and inhibitory receptors of the adenylate cyclase system are coupled to the cyclase catalytic unit through two GNPs, Gs and Gi, which mediate stimulation and inhibition, respectively. In addition, rhodopsin, the photon receptor in retinas, is coupled to its target enzyme, a cGMP phosphodiesterase, through a GNP known as transducin (T). Go, a GNP which interacts functionally with rhogopsin and muscarinic receptors, does not appear to be involved in adenylate cyclase regulation. All of these GNPs exhibit structural and functional similarities and are heterotrimers of Alpha, Beta, and Gamma Subunits. A cDNA clone, Lambda609, was isolated from a bovine retinal Lambdagt10 library using oligonucleotide probes complementary to reported sequences in two clones of the Alpha subunits of transducin TAlpha). Sequences of several tryptic peptides from bovine brain GoAlpha were identical to deduced amino acid sequences in Lambda609. Nucleotide and deduced amino acid sequences of Lambda609 also revealed significant similarities to corresponding regions of bovine TAlpha, GsAlpha, GiAlpha, and rat brain GoAlpha. Lambda609 encodes for an amino acid sequence highly homologous to the region surrounding the arginine residue that is ADP-ribosylated by choleragen in TAlpha as well as a sequence at the carboxy terminus which includes a cysteine residue at the position of the cysteine in TAlpha and GiAlpha that is the substrate for ADP-ribosylation by pertussis toxin. Northern analysis revealed that, of several tissues examined, the levels of RNA coding for GoAlpha are highest in the brain.